The proposed experiments are designed initially to investigate one aspect of that complex structure that makes up telomeres in higher eukaryotes--the DNA structure that permits complete replication of the ends of linear DNA molecules. 1. Molecular cloning by function. True telomeres will not attach to broken or restricted ends of DNA and thus shoud not be isolated by conventional procedures of recombinant DNA technology. We will use the linear yeast plasmid that permits cloning of any DNA segment able to function as a chromosomal terminus in yeast (Szostak and Blackburn, 1982, Cell 29:245). This system will be used to clone Drosophila DNA segments able to function as telomeres in yeast. Cloned segments will be identified as telomeres and their structure studied by variety of restriction enzyme and hybridization analyses. 2. Molecular cloning of telomere-like sequences. Drosophila DNA contains sequences with homology to the repeated CCCCAA tracts found in other eukaryotic telomeres. We will isolate segments having homology to these tracts by conventaional cloning in lambda. These procedures shoud not give an intact terminus but any clone that can be shown to be near -terminal will be an alternative probe for characterizing the extreme ends of the Drosophila chromosomes. These clones will also be used in alternative strategies for functional cloning if the yeast procedures does not succeed. 3. Transformation of Drosophila with telomere-containing transposons. The successful cloning of Drosophila telomeres will permit the construction of new chromosomal termini to study the function of each telomeric component. In yeast head-to-head fused telomeres (constructe in vitro) resolve into linear forms in vivo. Drosophila telomeres will be similarily fused in vitro, incorporated into transposons, and used to transform Drosophila embryos. If resolution occurs (or can be forced) we will be able to construct new telomeres with any desired set of sequences. Study of the genetic and cytological properties of such new chromosomal termini will provide experimental evidence on many of the propose functions of telomeres and telomeric heterochromatin.